PROCESS FOR PRODUCING 4-HYDROXY-CINNAMYL ALCOHOLS

Abstract: The invention is concerned with a process for preparing 4-hydroxy-cinnamyl alcohols by enzymatically oxidizing 4-allyl-phenols using vanillyl alcohol oxidase. Preferred substrates are eugenol and chavicol leading to coniferyl alcohol and coumaryl alcohol respectively. The vanillyl alcohol oxidase may be obtained from Penicillium simplicissimum.

Description: Process for producing 4-hydroxy-cinnamyl alcohols The invention concerns an enzymatic process for producing 4- hydroxy-cinnamyl alcohol and analogous compounds, hereinafter collectively referred to as 4-hydroxy-cinnamyl alcohols. More particularly the invention concerns an enzymatic process for converting 4-allyl-phenols to 4- hydroxy-cinnamyl alcohols. 4-Hydroxy-cinnamyl alcohols are important intermediates. Thus, 3-methoxy-4-hydroxy-cinnamyl alcohol (coniferyl alcohol) is an intermediate in the synthesis of vanillin which is one of the most widely used aroma components in the food industry. Likewise, 4-hydroxy-cinnamyl alcohol itself (coumaryl alcohol) may be converted to 4-hydroxybenzaldehyde which is also of interest in the flavour industry. Processes known for converting coniferyl alcohol to vanillin include direct conversion such as described in EP-A-0 542 348, or conversion to other intermediates followed by further conversion of those intermediates to vanillin. Thus, coniferyl alcohol may be converted chemically to coniferyl aldehyde. Coniferyl aldehyde in turn may be converted to vanillin using Arthrobacter globiformis mutants as described in DE-A-3604874. Furthermore, coniferyl alcohol may be converted to a suitable ester such as its benzoic acid ester, which in turn can be converted to vanillin, again as described in EP-A-0 542 348 4-Allyl-phenols may be obtained from various biological sources. Thus, eugenol is an abundantly available starting material e.g. obtained from clove leaf and clove bud oil. On the other hand, various microbiological processes are known for directly converting eugenol into vanillin, see e.g. processes mentioned in DE-A-3604874, using certain Corynebacterium or Pseudomonas species, and the process described in EP-A-0 405 197. However, these processes suffer from the disadvantage that they use inaccessible micro- organisms, give rise to many undesirable byproducts or give very low yields. Therefore, an efficient enzymatic or microbiological process for converting eugenol to coniferyl alcohol would be an important step towards an economical process for obtaining vanillin. It has now been found that various 4-hydroxy-cinnamyl alcohols can be prepared by enzymatic oxidation of the corresponding 4-allyl-phenol using vanillyl alcohol oxidase, an intracellular aryl alcohol oxidase having covalently bound FAD as prosthetic group. The enzymatic oxidation process according to the invention is depicted in the reaction scheme below: wherein Rl may be H, OH or an alkoxy group with up to 3 carbon atoms and R2 may be H or an alkyl group with up to 3 carbon atoms. Preferably Rl is H or OCH 3 . Preferably R2 is H. The compounds according to the general formula I for the purpose of this invention are collectively called 4-allyl- phenols, like the compounds of general formula II are collectively called 4-hydroxy-cinnamyl alcohols. The process is particularly useful for converting eugenol into coniferyl alcohol and chavicol in coumaryl alcohol. Vanillyl alcohol oxidase is induced intracellularly when Penicillium simplicissimu is grown on veratryl alcohol. Its isolation and characterization have recently been described in Eur. J. Biochem 208. 651-657 (1992). It was described therein to have a substrate specificity limited to vanillyl alcohol and 4-hydroxy-benzyl alcohol, and to oxidize neither other aromatic or non-aromatic alcohols, nor aromatic aldehydes. The process may be carried out using viable microbial cells containing the enzyme, dead cells, unpurified cell-free extracts or an enzym preparation obtained by subjecting the cell extract to one or more purification steps such as described in Eur. J. Biochem 208. 651-657 (1992). Preferably cells or a cell-free extract or enzym preparation obtained from P. simplicissimum grown on veratryl alcohol is used. Also, suitable enzyme preparations may be obtained from other microorganisms after they have been subjected to a genetic engineering process which enables them to make the vanillyl alcohol oxidase. In carrying out such genetic engineering process structural information on the enzyme in P. simplicissimum is used. The process is carried out in solvents usual for such microbial or enzymatic processes, particularly water, at a pH between 7 and 12, preferably between 8 and 11, more preferably between 9 and 11. The temperature of the reaction mixture is kept between 5 and 60°C, preferably between 15 and 45°C, more preferably between 20 and 40°C. When live cells are used, the 4-allyl-phenol concentration in the reaction mixture is chosen such as to be non-toxic for the cells, using fed-batch or similar techniques if desired. For dead cells or other enzyme preparations the substrate concentration is preferably chosen below 10 mM, more preferably below 5 mM. The oxygen concentration in the reaction mixture is preferably kept at a level to be non- limiting. The 4-hydroxy-cinnamyl alcohol may be isolated from the reaction mixture in ways known in the art such as by extraction with an organic solvent. The reaction may be carried out in a suitable two phase system, preferably consisting of an aqueous phase in which the reaction takes place (the reaction phase) and an organic phase chosen such that the reaction product accumulates in the organic phase. Alternatively, the non-reaction phase may be a solid absorbent which absorbs the reaction product from the reaction phase. EXAMPLE To 27 μmol eugenol in 7,5 ml 50 mM glycine/NaOH buffer (pH 10,0) was added 1 unit vanillyl alcohol oxidase. The mixture was kept saturated with air and incubated for 3 hours at 35°C. Thereafter the mixture was extracted twice with dichloromethane. The extract was evaporated and the coniferyl alcohol content in the residue analysed gas chromatographically. Eugenol had been converted quantitatively to coniferyl alcohol. The example was repeated using Chavicol as the substrate. Likewise, it was converted quantitatively to coumaryl alcohol. 1 unit vanillyl alcohol oxidase is the amount of enzyme necessary to oxidize 1 μ ol vanillyl alcohol in 1 minute at 30°C and pH 10,0.

Claims: CLAIMS 1. Process for the preparing 4-hydroxy-cinnamyl alcohols characterized in that a 4-allyl-phenol according to general formula I in the reaction scheme wherein Rl may be H, OH or an alkoxy group with up to 3 carbon atoms and R2 may be H or an alkyl group with up to 3 carbon atoms, is enzymatically oxidized using vanillyl alcohol oxidase to obtain a 4-hydroxy-cinnamyl alcohol according to general formula II wherein Rl and R2 have the meaning given above.. 2. Process according to claim 1 characterized in that R2 is H. 3. Process according to claims 1 or 2 characterized in that Rl is H or OCH 3 . 4. Process according to any one of claims 1-3 characterized in that the oxidation reaction is carried out at pH between 7 and 12. 5. Process according to any one of claims 1-4 characterized in that the oxidation reaction is carried out between 5 and 60°C. 6. Process according to any one of claims 1-5 characterized in that the enzyme is obtained from Penicillium simplicissimum 7. Process according to any one of claims 6 characterized in that the enzyme is obtained from P. simplicissimum grown on veratryl alcohol. 8. Process according to any one of claims 1-7 characterized in that the enzyme is present in viable microbial cells. 9. Process according to any one of claims 1-7 characterized in that the enzyme is present in dead cells, a cell-free extract or an enzyme preparation prepared therefrom.

Inventors: DE JONG EDSERD, FRAAIJE MARCO WILHELMUS, VAN BERKEL WILHELMUS JOHANNES, DE JONG, EDSERD, FRAAIJE, MARCO, WILHELMUS, VAN BERKEL, WILHELMUS, JOHANNES, H.
Applicants: BERKEL WILHELMUS JOHANNES H VA, FRAAIJE MARCO WILHELMUS, JONG EDSERD DE, QUEST INT, DE JONG, EDSERD, FRAAIJE, MARCO, WILHELMUS, QUEST INTERNATIONAL B.V., VAN BERKEL, WILHELMUS, JOHANNES, H.
Application year: 1994
Main classification: C12P7
IPC codes: C07C39, C12P7